SIRT1 Regulates Late Stage of Autophagy and Mitophagy via Autophagosome–Lysosome Fusion in Cardiomyocytes

Abstract

Sirtuin‐1 (SIRT1) is an NAD+‐dependent deacetylase implicated in autophagosome formation; however, whether SIRT1 also regulates autophagosome clearance during late‐stage autophagy remains unclear. Here, we investigated the role of SIRT1 in autophagosome clearance during autophagy and mitophagy in cardiomyocytes. Mitochondrial stress induced by carbonyl cyanide m‐chlorophenyl hydrazone (CCCP) decreased mitochondrial protein levels and increased phosphorylation of ubiquitin, a PINK1 target, in H9c2 cardiomyocytes. These CCCP‐induced decreases in mitochondrial proteins were prevented by co‐treatment with chloroquine, an inhibitor of lysosomal degradation, supporting the induction of CCCP‐triggered mitophagy. SIRT1 knockdown similarly prevented the CCCP‐induced reduction in mitochondrial proteins and led to the accumulation of autophagosomes containing fragmented mitochondria without attenuating ubiquitin phosphorylation, suggesting that SIRT1 acts downstream of mitochondrial tagging. Tandem GFP–RFP LC3 assay and LC3–LAMP1 colocalization analysis demonstrated impaired autophagosome–lysosome fusion following SIRT1 knockdown. In vivo, cardiomyocyte‐specific SIRT1 knockout mice exhibited elevated basal LC3‐II levels and a blunted LC3‐II response to chloroquine, consistent with impaired autophagic flux. In a doxorubicin (DOX)‐treated model, SIRT1 deficiency attenuated autophagosome degradation during the early period after DOX administration. Mechanistically, SIRT1 interacted with Rab7, a key regulator of autophagosome–lysosome fusion, raising the possibility that SIRT1 might regulate fusion through post‐translational modification of Rab7 or related components. Collectively, these findings identify SIRT1 as a regulator of autophagosome–lysosome fusion that promotes autophagosome degradation during autophagy and mitophagy in cardiomyocytes.